3iip

Evolutionary optimization of computationally designed enzymes: Kemp eliminases of the KE07 series
(see also Directed evolution, 2rkx, 3iio, and 3iiv)



A series of computationally designed enzymes that catalyze the Kemp elimination have described. Kemp eliminase (KE07) has TIM barrel scaffold. The Kemp elimination of 5-nitrobenzisoxazole was chosen as a model reaction for proton (H) transfer from carbon, simultaneously with the cut of the nitrogen–oxygen (N-O) bond, resulting in cyanophenol product. Such reaction is a critical step in many enzymatic reactions. The catalytic base (E101), the general acid/H-bond donor (K222), and the stacking residue (W50) make interactions with the 5-nitrobenzisoxazole at the active site of KE07. Directed evolution can significantly improve the stability, expression and activity of enzymes. In the catalytically improved directed evolutionary variants of KE07 containing the Ile7Asp mutation, Asp7 breaks the Glu101–Lys222 salt bridge (for example 3iiv, chain A is shown).

The comparison of the designed structure (colored orange) modelled in the presence of the 5-nitrobenzisoxazole and unbound crystal structure (2rkx, colored lime) of KE07 shows only limited conformational changes. In the designed structure, the amino group of Lys222 is ~4 Å away from the transition state phenolic oxygen, to stabilize the negative charge of the product phenoxide. However, Lys222 can also form a weak salt bridge with the catalytic Glu101 with a distance of ~3.6 Å. In the crystal structure of the KE07 without ligand (2rkx), the Glu101–Lys222 distance is 2.84 Å, i.g. within salt bridge distance. This is probably a unique feature of the unbound conformation of KE07. As was mentioned above, in the catalytically improved directed evolutionary mutants of KE07 bearing the Ile7Asp mutation, Asp7 breaks the Glu101–Lys222 salt bridge (in the evolved mutants, the NεLys222–OγGlu101 distance is 3.3–5.7 Å), in some cases directly (as in the present case 3iiv, chain A) interacting with Lys222 (the NεLys222–OβAsp7 distance is 2.8–5.7 Å). An overlap of the structures of the wildtype (lime) KE07 and the its <font color='lightskyblue'>evolved Ile7Asp mutant reveals how the Ile7Asp mutation causes the shift of the Lys222 side chain away from Glu101.

The residues Arg5, Glu46, Lys99, and Glu167 of <font color='lime'>KE07 unbound wildtype crystal structure (2rkx) form <scene name='3iio/Ali1/1'>electrostatic network at the bottom of the active site. In this case Lys222, of course, is not involved in this network, because it could not form electrostatic interaction with Ile7. Ile7Asp mutation in the evolved mutants introduces Lys222 to this electrostatic network or <scene name='3iio/Ali1/3'>directly as <font color='lightskyblue'>in case of 3iiv<font color='lightskyblue'>, chain A, or <scene name='3iio/Ali1/4'>via water molecule (3iiv, <font color='tan'>chain B ).

The crystal structures of the catalytically improved directed evolutionary KE07 mutants also demonstrate that replacement of side chains via mutations, combined with minor backbone changes, could allowed the new enzyme–substrate interactions. For example, <scene name='3iio/Ali1/5'>superposition of the structures of the <font color='orange'>KE07 design and evolved <font color='darkmagenta'>KE07 round 4 1E/11H chain A (3iio) reveals that the mutation Gly202Arg caused a shift of the adjacent loop (residues 175–177) and could introduce a new interaction with the nitro group of the 5-nitrobenzisoxazole. The directed evolution also creates new interaction networks of charged surface residues at the upper part of the active site. In the <scene name='3iio/Ali1/6'>wildtype KE07 (2rkx, <font color='lime'>colored lime ), Gly is in the position 202, Asn is in the position 224, and distance between Asn224 O and His201 N is 7.9 Å. In the evolved variants, following the Gly202Arg and Asn224Asp mutations, Asp224 and His201 gradually became closer, with distances between Asn224 O and His201 N of 4.6 Å in the <scene name='3iio/Ali1/7'>round 4 variant (3iio, <font color='darkmagenta'>colored darkmagenta ) and 3.6 Å in the <scene name='3iio/Ali1/8'>round 7 variant, chain B (3iiv, <font color='tan'>colored tan ). In rounds 6-7 variants, Asp224 can potentially interact with Arg202 and with His201. This network of Arg202–Asp224–His201 also brings His201 closer to the substrate (not shown). Interestingly, the <scene name='3iio/Ali1/9'>conformation of Trp50 at the active site in <font color='lightskyblue'>chain A of round 7 1/3H variant (3iiv) significantly differs from those in all other structures, including <font color='tan'>chain B within the asymmetric unit of round 7 1/3H. Of note, that Trp50 of chain A overlaps the substrate.

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About this Structure
3IIP is a 6 chains structure with sequences from Escherichia coli. Full crystallographic information is available from OCA.